Modification of the poly G-poly C complex by incorporation of adenosine in the purine chain

Antiviral and interferonogenic activity of the complexes of poly(G,A) . poly(C) and poly(G) . poly(C) was studied in mice and cell cultures. Three out of 4 complexes of poly(G,A) . poly(C) had insignificant antiviral and interferonogenic activity in chick embryo cells. One of the complexes induced low levels of interferon production in mice and decreased the rate of their death from experimental forest-spring encephalitis. The activity of poly(G) . poly(C) in the above cell systems was much more pronounced. Unlike this complex, some complexes of poly(G,A) . poly(C) showed a noticeable activity in the cells of Primates. The effect of the noncomplementary base in the purine thread of poly(G) . poly(C) on its biological activity and nucleotide composition is discussed.     

Effect of interferonogenic molecular complex of yeast RNA--tilorone on DNA, RNA and protein synthesis in vitro

In the experiments in vitro using the primary mononuclear cells (MNC) culture of the human peripheral blood the influence of interferonogenic yeast RNA-tilorone molecular complex on the DNA, RNA and protein synthesis was studied. The complex was shown to inhibit the insertion of 3H-thymidine, 3H-uridine and 3H-leucine into DNA, RNA and protein of MNC total pool (by 13, 1 and 40% respectively); that was practically conformed with this synthesis inhibition upon to a natural origin polynucleotide interferon inducers--lariphan (9, 0 and 57% respectively) and ridostin (9, 0 and 56% respectively) action, and at the same time rather less than poly(I)-poly(C) (14, 5 and 62% respectively). In the case of preliminary cell stimulation by the mitogen PHA the complex revealed comitogenic action at a concentration 25 micrograms/ml, that corresponded to optimal for interferonogenesis; the increase of the doses till 100-1000 micrograms/ml lead to in the reversal effect. To proceed from mutual relation between interferonogen preparations influence on the mentioned synthesis and their cytotoxicity the conclusion was about made the complex promising usage as an interferon inducer both in vitro and in vivo conditions.     

Para-aminobenzoic acid--an interferon inducer]

Para-aminobenzoic acid (PABA) was shown to be an early type interferon inductor. PABA (10 micrograms/ml) induced interferon production in vitro in the cells of human peripheral blood and in vivo in albino mice (10 mg/kg). The results of the study suggested that PABA was able to induce production of interferon-alpha/beta in various immunocyte populations. By its interferonogenic activity PABA was comparable with the known interferon inductors. One of the mechanisms of the previously described in vivo antiherpes action of PABA can be attributed to its interferon inducing activity.     

Live enteroviral vaccines for the emergency nonspecific prevention of mass respiratory diseases during fall-winter epidemics of influenza and acute respiratory diseases]

The results of the 3-year controlled trials of a new method of nonspecific urgent prophylaxis of influenza and acute respiratory diseases (ADR) by immunization of healthy adults with standard live enterovirus oral vaccines, introduced in 2-3 administrations at intervals of 7-10 days, at the initial stages of autumn and winter epidemics are presented. Observations, carried out in three republics, covered more than 150,000 persons immunized with enterovirus interferonogenic vaccines. A considerable decrease in morbidity rate among the vaccinees was achieved (on the average, by 3.2 times) in comparison to that among nonimmunized subjects. The method of nonspecific prophylaxis with live enterovirus interferonogenic vaccines is recommended during outbreaks of diseases induced simultaneously by several causative agents of influenza and ARD, as well as by pathogenic enterovirus strains.     

Use of interferon inducers in treating viral diseases

The results obtained in the experimental and clinical study of the preparations of mephenamine acid and levamisole with respect to their interferonogenic and interferon-stimulating action and their therapeutic effectiveness in influenza and virus hepatitis are presented. The study has revealed that mephenamine acid, along with its capacity for stimulating the processes of interferon formation in the body, produces a pronounced curative effect, prevents the development of postinfluenza complications much more effectively than levamisole and remantadin and accelerates the processes of reparation in virus hepatitis.     

Comparative antiviral and interferonogenic activity of natural and synthetic interferon inducers in vitro and in vivo]

Biological activities of the RNA replicative form of phage f2, a natural interferon inductor and poly-I -- poly-C, a synthetic polyribonucleotide complex were studied comparatively. Differences in the comparative interferonogenic and antiviral activity of the inductors were as dependent on the type of the cell system. It was shown that DEAE-dextran increased the interferon-inducing activity of RFf2 in the cell culture by 4 to 8 times. The dynamics of the interferonogenic and antiviral activity of RFf2 in the L-929 cell culture was studied. Interferon appeared in the culture fluid in 6--8 hours and reached its maximum titers (128 IU50/ml) by the 24th hour, the maximum protection of the cells being also developed by the 12th--24th hour, reaching on an average 51 g PFU/ml. It was shown in the experiments with green marmosets that administration of RFf2 in the form of aerosol in a dose of 2.3 mg/kg induced interferon production in the blood serum the titers of which amounted to 80--160 IU50/ml 24 hours after the administration.     

Mitogenic effect of double-stranded RNA in human fibroblasts: role of autogenous interferon

The synthetic double-stranded RNA polyinosinate-polycytidylate [poly(I).poly(C)] was mitogenic in cultures of human foreskin fibroblasts, as demonstrated by a stimulation of 3H-thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)-beta in human fibroblasts. Single-stranded poly(l) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)-beta, added to poly(I).poly(C)-stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN-beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN-beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)-stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN-beta can exert negative feedback control of cell proliferation.     

Immunobiological properties of staphylococcal enterotoxins type A, B, C, D and E

Comparative study of mitogenic and interferonogenic properties of staphylococcal enterotoxins of different serotypes is done. It is revealed that preparations of enterotoxins are polyclonal mitogens and have interferon-inducing activity. It is stated that enterotoxin of D type has the highest mitogenic activity, which is shown by interferon-inducing activity of A type toxin.     

A simple method of purifying staphylococcal alpha-toxin and a study of its properties]

The simple method is proposed for isolation and purification of staphylococcal alpha-toxin that permits one to obtain the homogeneous toxic protein with high activity. The time necessary for maximal toxin production at cultivation has been defined. The thermostability and interferonogenic characteristics of the obtained alpha-toxin were studied.     

Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid.

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Nature and possible origin of Human Serum ribonuclease

Human Serum contains an acidic RNase which is glycoprotein in nature. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. Its activity either on poly (C) or RNA is endonucleolytic and is absolutely dependent on citrate or phosphate. It exhibits highest preference for the secondary phosphate esters of cytidine 3′-phosphates. It has no action on cytidine 2′:3′-cyclic phosphate. Poly (A) and poly (G) are not only refractory to its action, but also inhibit its action on poly (C). Its rate of hydrolysis of Poly (U) is about 2% of that of poly (C). It differs from bovine pancreatic RNase. It is, however, similar to human pancreatic RNase suggesting that its primary source is pancreas.     

Purification and Properties of a Ribonuclease in Human Urine that Hydrolyses Polycytidylic Acid

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylaraide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is aci c and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21, 500.

It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3′-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2′: 3′-cyclic phosphate or uridine 2′:3′-cyclic phosphate.

Its rate of hydrolysis of poly (U) is less than 2% of that of poly (C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U).

It differs from bovine pancreatic RNase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Interferonogenic activity of immobilized ribopolynucleotides in vitro

In vitro experiments the authors have studied a property of yeast RNA--tilorone hydrochloride complex covalently linked to spheron to induce the synthesis of interferons type I (alpha- and beta-interferons) in the culture of peripheral mononuclear human cells. Such a complex is shown to possess a marked interferonogenic activity. The data obtained appear to be a proof of the interferon induction to be realised by a mechanism needing at the first stage the contact between the inducer and the cell surface without its penetration into the cell.     

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.     

Comparative cytotoxic characteristics of the interferon-inducing yeast RNA-tilorone complex]

Cytotoxicity of the yeast RNA-tilorona molecular complex (MC) with interferonogenic properties and its influence on the DNA replicative synthesis were studied in experiments with human lymphocytes and 3 cell lines. It was shown that the MC doses of 25, 100 and 250 micrograms/ml were absolutely nontoxic for all the cell lines. The main parameters of the MC toxicity based on the cell viability were calculated. The parameters were found to correlate in the order of their magnitude with those relating to interferonogens of the polynucleotide nature. Within the dose ranges of 10 to 100 micrograms/ml the MC had a stimulating effect on replicative processes in the cells. It was concluded that the use of the MC as an inductor in large-scale manufacture of human and animal interferons of type 1 was promising.     

Human granulocyte ribonuclease.

Human granulocytes contain an RNase which is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for the secondary phosphate esters of uridine 3′-phosphates. It has no action on uridine 2′: 3′-cyclic phosphates. Poly (A) and poly (G) are inert to its action. Its rate of hydrolysis of poly (C) is about 1% of that of poly (U). It differs from bovine pancreatic RNase and human serum RNase. Because of its unique specificity, this enzyme might serve as a biochemical marker in certain granulocyte disorders.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Induction of histamine secretion by polycations

Poly(arginine), poly(lysine) and poly(ornithine) induce histamine secretion from human basophil leukocytes in the concentration range 1--100 nmol/l. Histamine secretion induced by poly(arginine) requires extracellular calcium at 0.1--1 mmol/l. Strontium (1--10 mmol/l) will substitute for calcium. Lanthanum (30--90 nmol/l) inhibits histamine release induced by poly(arginine). Histamine secretion induced by poly(arginine) is inhibited by 1--30 mumol/l N-ethyl-maleimide, 0.3--3 mmol/l 2-deoxy-D-glucose, 0.3--3 mmol/l dibutyryl cyclic AMP, 0.3--3 mmol/l, adenosine 3'5'-cyclicphosphorothioate. The action of poly(arginine) is inhibited by pretreatment of basophils at 47 degrees C or with neuraminidase. 10 microgram/ml heparin inhibits the response to poly(arginine). Histamine releasing potency of the polymer amino acids is dependent on chain length of the peptide. Succinylated poly(lysine) is inactive. Monomer amino acids do not release histamine and do not inhibit the action of the polymers. Histones and protamine do not release histamine, nor do the peptides eledoisin and tuftsin. Putrescine, cadaverine, spermine and spermidine do not release histamine. Poly(glutamic acid), poly(aspartic acid) and poly(tyrosine) are also inactive. The IgE-mediated release of histamine appears to be independent of that mediated by poly(arginine).     

Mechanism of adjuvant activity of poly A:U on antibody responses to hapten-carrier conjugates

The adjuvant action of poly A:U has been analyzed in a system measuring humoral immune responses to hapten-carrier conjugates in mice. Administration of poly A:U at the time of primary immunization with 2,4-dinitrophenyl (DNP)-keyhole limpet hemocyanin (KLH) shortens the induction period for, and heightens the magnitude of peak anti-DNP antibody and specific memory cell production. In order to define the cellular locus of poly A:U action, the effect of this adjuvant on adoptive secondary anti-DNP antibody responses was studied. Spleen cells from DNP-KLH-primed donors, which normally fail to develop adoptive secondary anti-DNP responses to a heterologous conjugate such as DNP-bovine gamma globulin (BGG), can be stimulated to do so when an appropriate dose of poly A:U is administered with DNP-BGG. The capacity for poly A:U to exert this effect requires the presence of T lymphocytes, since depletion of such cells by treatment of the donor cell inoculum with anti-θ serum and complement in vitro prior to adoptive transfer abrogates the response to DNP-BGG plus poly A:U. Moreover, evidence is presented that demonstrates that poly A:U exerts its adjuvant action on the small number of unimmunized BGG-specific T lymphocytes in the donor cell inoculum. This conclusion derives from the failure of poly A:U to augment adoptive secondary anti-DNP responses to the DNP derivative of a nonimmunogenic copolymer of d-glutamic acid and d-lysine (d-GL) for which there are few or no specific, functional T cells.     

Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.